SUMMARY Toxoplasma gondii is a protozoan parasite that is responsible for encephalitis in people with AIDS. Clinical disease is due to unchecked proliferation of tachyzoite forms that replicate rapidly within nearly all nucleated cells. When infection is controlled by the immune system, tachyzoites differentiate into bradyzoites that persist within tissue cysts. Bradyzoites may later reactivate and cause clinical disease in immunocompromised individuals including people living with HIV. During T. gondii replication within host cells, the parasite implements a coordinated pattern of sequential gene expression. In response to stress, the parasite alters its metabolism, surface antigens, and cell cycle to transition from tachyzoite to bradyzoite. Bradyzoites persist for the lifetime of an infected host and are refractory to treatments that eliminate the tachyzoites. cAMP signaling via cAMP-dependent kinase (PKA) is a conserved pathway that regulates metabolism and stress responses in most eukaryotes. Using the CRISPR/Cas9 system, we disrupted PKAc3, one of the isoforms of the catalytic subunit of the T. gondii PKA, and showed that it is a negative regulator of the tachyzoite-bradyzoite transition. Parasite strains lacking PKAc3 have constitutive differentiation to bradyzoite forms. We hypothesize that cAMP signaling leads to phosphorylation of substrates by PKAc3, promoting tachyzoite growth and repression of bradyzoite genes. We propose that the major mechanism for PKAc3 regulation of the bradyzoite program is via modulation of tachyzoite chromatin accessibility and regulation of the activity of repressor(s) of bradyzoite gene expression. Therefore, we plan to identify substrates of PKAc3 that act as transcriptional repressors and chromatin structure modifiers that regulate the bradyzoite developmental program using a combined proteomics and genetics approach. We will use comparative phosphoproteomics to identify candidate PKAc3 substrates. In addition, we will compare the phophoproteome and gene expression of Type I and Type II parasites and mutants lacking PKAc3, to determine if we can identify differences in gene expression or phosphorylation patterns that provide a mechanism for the differential ability of Type I and Type II strains to differentiate into bradyzoite forms. Finally, to link gene expression to PKAc3 activity, we will test the hypothesis that candidate chromatin factors and AP2 factors regulate bradyzoite gene expression in a PKAc3 dependent manner.